Relative Contribution of Nedd4 and Nedd4-2 to ENaC Regulation in Epithelia Determined by RNA Interference

Abstract

Epithelial Na؉ transport is regulated in large part by mechanisms that control expression of the epithelial Na ؉ channel (ENaC) at the cell surface. Nedd4 and Nedd4-2 are candidates to control ENaC surface expression, but it is not known which of these proteins contributes to ENaC regulation in epithelia. To address this question, we used RNA interference to selectively reduce expression of Nedd4 or Nedd4-2. We found that endogenous Nedd4-2, but not Nedd4, negatively regulates ENaC in two epithelial cell lines (Fischer rat thyroid and H441); small interfering RNA (siRNA) against Nedd4-2 increased amiloride-sensitive Na ؉ current (compared with control siRNA), but Nedd4 siRNA did not. A mutation associated with Liddle's syndrome (␤ R566X ) abolished the effect of Nedd4-2 siRNA, suggesting that a defect in ENaC regulation by Nedd4-2 contributes to the pathogenesis of this inherited form of hypertension. Previous work found that Nedd4-2 is phosphorylated by serum and glucocorticoid-regulated kinase, a Ser/Thr kinase induced by steroid hormones. Here we found that Nedd4-2 phosphorylation contributes to ENaC regulation by steroid hormones. Consistent with this model, ENaC stimulation by dexamethasone was reduced by Nedd4-2 siRNA and by overexpression of a mutant Nedd4-2 lacking serum and glucocorticoid-regulated kinase phosphorylation sites. Thus, endogenous Nedd4-2 negatively regulates ENaC in epithelia and is a component of a signaling pathway by which steroid hormones regulate ENaC. Defects in this regulation may contribute to the pathogenesis of hypertension.The rate of Na ϩ transport across a variety of epithelia (e.g. kidney-collecting duct, airway, colon) is regulated in large part by mechanisms that control expression of the epithelial Na1 at the apical cell surface. ENaC, a heteromultimeric complex of three homologous subunits (␣-, ␤-, and ␥-ENaC), forms a pathway for Na ϩ to enter the cell at the apical membrane followed by Na ϩ exit at the basolateral membrane mediated by the Na ϩ -K ϩ -ATPase (1, 2). Defects in ENaC regulation cause blood pressure abnormalities (hypertension or hypotension) (3) and contribute to the pathogenesis of cystic fibrosis (4).Although regulation of ENaC surface expression is critical for Na ϩ homeostasis, little is known about the mechanisms responsible. Previous work (reviewed in Ref. 5) identified Nedd4 and Nedd4-2, HECT WW domain ubiquitin-protein ligases, as candidates. First, Nedd4 and Nedd4-2 are capable of binding to ENaC; multiple WW domains bind to PY motifs located in the C terminus of each ENaC subunit (6 -10). Importantly, mutation of these PY motifs causes an inherited form of hypertension (Liddle's syndrome) (11,12). Second, Nedd4 and Nedd4-2 inhibit ENaC when coexpressed in heterologous cells; a ubiquitin ligase domain catalyzes ENaC ubiquitination, targeting the channel for degradation (9,13,14). As a result, reduced ENaC surface expression decreases Na ϩ current. However, an important limitation is that previous studies tested the effect of Nedd4 and Nedd4-2 over...

cAMP and Serum and Glucocorticoid-inducible Kinase (SGK) Regulate the Epithelial Na+ Channel through Convergent Phosphorylation of Nedd4-2

“…FRT epithelia express endogenous Nedd4-2 (17) and reconstitute cAMP regulation of ENaC (20). Our strategy was to decrease Nedd4-2 expression using RNA interference; in previous work, we generated siRNAs that selectively silence Nedd4-2 or Nedd4 (a related E3 ubiquitinprotein ligase) (17). Transfection of FRT epithelia with ENaC generated amiloride-sensitive Na ϩ current that was stimulated by cAMP agonists ( Fig.…”

“…SGK K127M was described previously (15). Small interfering RNAs (siRNA) were generated and characterized previously (17). Peptides (15 amino acids) corresponding to Ser-221 (biotin-EPSSRLR-SCSVTDAV), Thr-246 (biotin-APAGRARSSTVTGGE), and Ser-327 (biotin-PQIRRPRSLSSPTVT) were synthesized (Howard Hughes Medical Institute/Keck Facility, Yale University) as well as equivalent peptides in which Ser-221, Thr-246, and Ser-327 were mutated to alanine.…”

“…Similar to Liddle's syndrome, decreased binding leads to increased ENaC expression at the cell surface and, hence, increased Na ϩ transport. Importantly, this mechanism contributes to ENaC regulation by aldosterone and glucocorticoids (17), which induce SGK transcription (18,19).…”

Ankyrin G Expression Regulates Apical Delivery of the Epithelial Sodium Channel (ENaC)

“…As mentioned in the introduction, ubiquitylation and internalization of ENaC is a well established means of reducing the number of ENaCs at the plasma membrane (12,51). We therefore tested whether AnkG overexpression reduced ENaC internalization rates.…”

Neural Precursor Cell-expressed Developmentally Down-regulated Protein 4-2 (Nedd4-2) Regulation by 14-3-3 Protein Binding at Canonical Serum and Glucocorticoid Kinase 1 (SGK1) Phosphorylation Sites

“…2 a member of the homology to the E6-associated protein C terminus (HECT) family of E3 ubiquitin ligases (1,2), is a physiologically important regulator of the epithelial Na ϩ channel (ENaC) (1,3). ENaC comprises ␣, ␤, and ␥ subunits and reabsorbs Na ϩ across several epithelia, including the distal nephron (4).…”